Attachment of cytotoxic T-lymphocytes (CTL) to Ag-bearing target cells (TC) results in the activation of CTL and in the destruction of TC. Molecular mechanisms of CTL-TC interactions are poorly understood. We attempted to identify proteins and enzymatic activities involved in CTL activation and effector functions. Several independent complementary approaches were used to address the questions. I. We found that protein kinase C activators and Ca++ ionophores bypas the requirement for Ag-receptor mediated recongnition by triggering conjugate formation and delivery of a lethal hit to non-Ag-bering TC by murine CTL. These results appear to rule out the role of links between antigen and antigen receptor in conjugate formation and CTL activation and implicate protein kinase C and calcium increases in these CTL functions. II. In an attempt to clarify Ca++-dependent reactions we have studied Ca++-binding and Calmodulin (CaM)-binding proteins in lympocytes in association with Dr. R. Kincaid. We found that the major CaM-binding protein in resting T- and B-lymphocytes is a protein phosphatase, "calcineurin". This result suggests the importance of reactions of dephosphorylation in "on" and "off" signalling in lymphocytes. III. We have demonstrated that "on" signals in CTL clones result in the exocytosis of the content of low pH intracellular granules and that this exocytosis is also regulated by protein kinase C and celcium. We have proposed a new general short term assay for the study of CTL activation. This assay is based on the detection of activity of a serine esterase, which is secreted rapidly inthe supernatants in response to antigen-receptor crosslinking.